PROJECT SUMMARY Extracellular matrix (ECM) components, including fibulin-4, play critical roles in aortic aneurysm formation, either directly through genetic mutation or indirectly through mutations in non-ECM molecules, resulting in ECM disruption. Fibulin-4 is essential for elastic fiber assembly: however, additional functions of fibulin-4 in the vasculature have not been investigated. Fibulin-4 deficient aortas show increased cell proliferation prior to and during aneurysm formation, but the origin of these cells and mechanism(s) driving their activation are unknown. Furthermore, the observed proliferation is not as widespread as expected if loss of elastic fiber integrity were the sole factor driving proliferation. This suggests that additional localized changes in the ECM microenvironment are necessary. Our long-term objective is to better understand the molecular mechanisms leading to aortic aneurysms and dissections so that treatments can be developed. Specifically, our goal is to identify the subpopulations of proliferative cells and the specific molecular defects in collagen that result from fibulin-4 deficiency. Our preliminary data show deficient collagen cross-linking and altered expression of collagen types in fibulin-4 knockout mice. Mature fibrillar collagen inhibits cell proliferation in vitro: therefore, whether defects in collagen induced by fibulin-4 loss promote this increase in proliferation warrants investigation. The hypothesis we are testing is that fibulin-4 loss creates a microenvironment that promotes localized cell proliferation in the aortic wall, in part through disruption of collagen synthesis and maturation. We will use three separate fibulin-4 deficient mouse models to characterize the identity and determine the origin of the proliferating cells. Collagen synthesis, procollagen processing, and collagen maturation will be analyzed both in vitro in a mouse embryonic fibroblast (MEF) model system, and in whole aortic tissue. The MEF model will also be used to determine a causal link between collagen defects and the observed proliferation. Completion of these studies will allow us to define previously undescribed functions of fibulin-4 and better understand how changes in ECM create a permissive environment for proliferation in the vessel wall, leading to aneurysm formation. Additionally, cells with progenitor-like properties have been identified in the aorta, raising the question of whether the proliferating cells are progenitor cells. Finally, determining novel roles for fibulin-4 may lead to identification of new drug targets, which could potentially be used to delay or prevent aneurysm progression.